Juvenile Visceral Steatosis Mice Research Articles

Loss of wild-type carrier-mediated L-carnitine transport activity in hepatocytes of juvenile visceral steatosis mice.

Juvenile visceral steatosis (JVS) mice, which show systemic L-carnitine deficiency, may be an animal model of Reye's syndrome because of its phenotype of fat deposition and mitochondrial abnormalities in the liver. In this study, we compared the characteristics of the L-carnitine transport in isolated hepatocytes from wild-type and JVS mice. The uptake of L-carnitine by wild-type hepatocytes was saturable and the Eadie-Hofstee plot showed 2 distinct components. The apparent Michaelis constant (K(m)) and the maximum transport rate (V(max)) were 4.6 micromol/L and 59.5 pmol/15 min/10(6) cells, respectively, for the high-affinity component, and 404 micromol/L and 713 pmol/15 min/10(6) cells, respectively, for the low-affinity component. The high-affinity L-carnitine uptake occurred via an active carrier-mediated transport mechanism, which is characterized by Na(+)-, energy-, and pH-dependency. On the other hand, the high-affinity uptake was absent in JVS hepatocytes, and the values of K(m) and V(max) for the low-affinity uptake were 475 micromol/L and 557 pmol/15 min/10(6) cells, respectively. The hepatic carnitine transport properties in wild-type hepatocytes were similar to those of high-affinity mouse Octn2-transfected HEK293 cells. This study suggests that Octn2-type carnitine transporter is dysfunctional in hepatocytes of JVS mice.

Dysfunctions of the epididymis as a result of primary carnitine deficiency in juvenile visceral steatosis mice

The juvenile visceral steatosis mutant mice serve as an animal model of primary carnitine deficiency, classified as the sudden infant death syndrome. The defect in carnitine uptake was recently found to be due to a defect in the carnitine transporter gene. We herein report, for the first time, the characteristics of epididymal dysfunction in juvenile visceral steatosis mice. At 8–9 weeks of age, the epididymis was deformed and weight was significantly increased. Histologically, the duct of the proximal epididymis was dilated due to the accumulation of an unusually high level of spermatozoa. Spermatozoa were extravasated from the epididymal duct into the stroma. In contrast, the duct of the distal epididymis was constricted and contained no spermatozoa. Thus, the epididymal disorder causes obstructive azoospermia, leading to infertility.

Catecholamine metabolism inhibitors and receptor blockades only partially suppress cardiac hypertrophy of juvenile visceral steatosis mice with systemic carnitine deficiency

To clarify the mechanism of cardiac hypertrophy in carnitine-deficient JVS mice, we studied die possible role of catecholamine metabolism. Cardiac hypertrophy occurs 2 weeks after birth. The turnover of norepinephrine in the ventricles of JVS mice at 2 weeks was 3 times that of control, but it was not different from control at 5 days when the heart weight was not changed. To evaluate the accelerated norepinephrine turnover, we examined the effects of catecholamine metabolism inhibitors (α-methyltyrosine and 6-hydroxydopamine) and catecholamine receptor blockades (propranolol, prazosin and yohimbine) on the ratio of heart weight to body weight ( HW BW ) and on the augmented expression of atrial natriuretic peptide (ANP) and the down-regulated carnitine deficiency-associated gene expressed in ventricle (CDV-1). The HW BW ratio in JVS mice treated with catecholamine metabolism inhibitors and receptor blockades was significantly lower than in JVS mice without treatment, but still higher than in controls treated with each drug and in JVS mice treated with carnitine. The HW BW ratio of JVS mice with propranolol was not significantly different from that of JVS mice treated with catecholamine metabolism inhibitors and was significantly lower than that of JVS mice treated with prazosin and yohimbine. Northern blot analysis showed that the altered expression of ANP and CDV-1 was not corrected in the ventricles of JVS mice treated with any of the drugs except carnitine. These results suggest that the catecholamine metabolism accelerated in JVS mice ventricles at 2 weeks is not the major cause of cardiac hypertrophy, but probably promotes cardiac hypertrophy mainly through the β-adrenergic signaling pathway. The aberrant gene expression of ANP and CDV-1 found in JVS mice seems to be independent of catecholamine metabolism, and mediated primarily by the systemic carnitine deficiency.

Cardiomegaly in the juvenile visceral steatosis (JVS) mouse is reduced with acute elevation of heart short-chain acyl-carnitine level after l-carnitine injection

The long-term administration of l-carnitine was very effective in preventing cardiomegaly in juvenile visceral steatosis (JVS) mice, which was confirmed by heart weight as well as the lipid contents in heart tissue. After i.p. injection of l-carnitine, the concentration of free carnitine in heart remained constant, although serum free carnitine level increased up to 80-fold. On the other hand, a significant increase in short-chain acyl-carnitine level in heart was observed. These results suggest that increased levels of short-chain acyl-carnitine, not free carnitine, might be a key compound in the protective effect of l-carnitine administration in JVS mice.

Pyruvate dehydrogenase kinase 4 mRNA is increased in the hypertrophied ventricles of carnitine-deficient juvenile visceral steatosis (JVS) mice.

We isolated a mouse homologue cDNA of pyruvate dehydrogenase (PDH) kinase 4 (PDK4) with differential mRNA display as an up-regulated gene in the hypertrophied ventricles of juvenile visceral steatosis (JVS) mice with systemic carnitine deficiency. The PDK4 mRNA level was 5 times higher in JVS mice than in control mice under fed conditions. After 24 h starvation, this level increased to 20 times in JVS and 7 times in control, compared with the control fed level. On the other hand, carnitine administration reduced the high level of PDK4 mRNA in JVS mice to the control fed level. In control mice, the change in PDK4 mRNA was inversely correlated with the change in PDH activity. In JVS mice, however, the PDK4 mRNA level was not always correlated with the active-form PDH level.

A Missense Mutation of Mouse OCTN2, a Sodium-Dependent Carnitine Cotransporter, in the Juvenile Visceral Steatosis Mouse

Carnitine is an essential cofactor for the mitochondrial β-oxidation of long-chain fatty acids. The juvenile visceral steatosis (JVS) mouse, an animal model of systemic carnitine deficiency, is inherited in an autosomal recessive manner. Recently, a human OCTN2 gene encoding a sodium-dependent carnitine cotransporter was isolated and mapped to human chromosome 5q31. Since the mouse jvs locus was assigned to the region of chromosome 11 where it is syntenic to human chromosome 5q31, we isolated the mouse octn2 gene and screened for its mutation in the jvs mouse. DNA sequencing analysis disclosed a missense mutation from CTG (Leu) to CGG (Arg) at codon 352 located within the sixth transmembrane domain of octn2. This amino acid replacement possibly causes the conformational change of the protein that leads to dysfunction of the gene product. Hence, we conclude that octn2 is a candidate gene responsible for the JVS mouse.

Gene-Dose Effect on Carnitine Transport Activity in Embryonic Fibroblasts of JVS Mice as a Model of Human Carnitine Transporter Deficiency

Recently, the marked decline in renal carnitine reabsorption has been thought to account for the systemic carnitine deficiency in juvenile visceral steatosis (JVS) mice. We have conducted a kinetic analysis using embryonic fibroblasts derived from normal, heterozygous, and homozygous jvs mice and found that the high-affinity carnitine transporter ( K m = 5.5 μM), which shows Na + and temperature dependency and stereospecificity, is defective in homozygous jvs mice. Moreover, a gene dose-dependent decrease of carnitine transport activity, which was due to a decrease in the number of the transporter molecules, was found in heterozygous jvs mice. Similar phenomena have been observed in human primary carnitine deficiency. Therefore, JVS mice may be useful for understanding this extremely rare human hereditary disorder.

Characteristics of cardiac hypertrophy in the juvenile visceral steatosis mouse with systemic carnitine deficiency.

The juvenile visceral steatosis (JVS) mouse exhibits hereditary systemic carnitine deficiency and develops cardiac hypertrophy. The aim of this study was to clarify the characteristics of cardiac hypertrophy in the JVS mouse. Total carnitine content in IVS mouse heart was about 10% of that of control mouse heart at 4 and 8 weeks of age. The heart weight/body weight ratio was bigger in JVS mice than that in control mice at 2 weeks of age, and this difference in ratio increased with age. The wall areas of both ventricles and septum in JVS mice were larger than those of the control mice at 2 and 8 weeks. The myocyte diameter in both ventricular walls and septum in JVS mice was longer than that of the control mice. On electron microscopy, the percent of mitochondria in the myocyte was 66% in JVS mice, and 37% in control mice. The percent of lipid fraction in JVS mice was six-fold higher than that in control mice. Total content of adenine nucleotides in JVS mouse heart was about 60% of that in control mouse heart. Adenylate energy charge in JVS mouse heart was 63 and 45% of that in the control mouse heart at 4 and 8 weeks, respectively. Overall, the cardiac enlargement observed in this animal model could be accounted for by a proportional increase in the myocyte diameter in the ventricles and septum, accompanied by an increase in mitochondria. Furthermore, this cellular growth is associated with decreases in the levels of ATP and ADP, and adenylate energy charge.

Secondary abnormality of carnitine biosynthesis results from carnitine reabsorptional system defect in juvenile visceral steatosis mice

We characterized the l-carnitine transport system which is defective in the kidney of juvenile visceral steatosis (JVS) mice by using kidney slices and carnitine-related compounds, and evaluated the influence of the transport defect on the biosynthetic pathway of carnitine. The JVS mouse transport system defect, calculated as the difference in the transport activity between control and JVS mice, was simulated in control by γ-butyrobetaine ( γ-BB) and acetyl l-carnitine. γ-BB hydroxylase activity in the liver of JVS mice was double that of control mice, but the hepatic level of γ-BB in JVS mice was lower than in control mice, suggesting that the conversion of γ-BB to carnitine is not activated in the liver of JVS mice. JVS mice showed higher fractional excretions not only of l-carnitine but also of γ-BB and acetyl l-carnitine than control mice, indicating disturbed reabsorption of γ-BB and acetyl l-carnitine. The disturbed reabsorption of γ-BB in JVS mice is consistent with the fact that the amount of urinary γ-BB in JVS mice was four times that of control. The sum of the concentrations of l-carnitine, acetyl l-carnitine and γ-BB in the urine of JVS mice was not significantly different from that of the control, suggesting no remarkable increase of biosynthesis of γ-BB and carnitine in JVS mice. All these findings suggest that the carnitine transport system plays a role in the transport of γ-BB and that carnitine deficiency is aggravated by the disturbed reabsorption of γ-BB in the kidney.

The effect of carnitine on ketogenesis in perfused livers from juvenile visceral steatosis mice with systemic carnitine deficiency.

Juvenile visceral steatosis (JVS) mice have been reported to have systemic carnitine deficiency, and the carnitine concentration in the liver of JVS mice was markedly lower than that of controls (11.6 +/- 2.6 versus 393.5 +/- 56.4 nmol/g of wet liver). To evaluate the role of carnitine in mitochondrial beta-oxidation in liver, we examined the effects of carnitine on ketogenesis in perfused liver from control and JVS mice. In control mice, ketogenesis was increased by the infusion of 0.3 mM oleate, but not by L-carnitine. In contrast, although ketogenesis in JVS mice was not increased by the infusion of oleate, it was increased 2.5-fold by the addition of 1000 microM L-carnitine. Addition of 50, 100, and 200 microM L-carnitine increased ketogenesis in a dose-dependent manner. The infusion of 0.3 mM octanoate or butyrate increased ketogenesis in a carnitine-independent fashion in both control and JVS mice. These findings suggest that endogenous long chain fatty acids from accumulated triglycerides may be used as substrates in the presence of carnitine in JVS mice. The relationship between ketogenesis and free carnitine concentration was examined in livers from JVS mice. Ketogenesis increased as free carnitine levels increased until concentrations exceeded about 100 nmol/g of wet liver (340 microM). The free carnitine concentration required for half-maximal ketone body production in liver of JVS mice was 45 microM (13 nmol/g of wet liver), which corresponds to a K(m) value of carnitine palmitoyltransferase I. We conclude that carnitine is a rate-limiting factor for beta-oxidation in liver only when the carnitine level in liver is very low.

A novel gene suppressed in the ventricle of carnitine-deficient juvenile visceral steatosis mice

In order to clarify the pathogenesis and pathophysiology of cardiac hypertrophy in carnitine-deficient juvenile visceral steatosis (JVS) mice, we performed mRNA differential display analysis with total RNA extracted from the ventricles of control and JVS mice at 14 days of age. We identified four up-regulated genes, two known and two unknown, and a novel down-regulated gene. Northern blot analysis with a novel cDNA probe derived from the down-regulated gene fragment 8A2 revealed three mRNA species of 1.1-, 1.3-, and 2.6-kb. The 1.1- and 1.3-kb mRNA species were found only in the heart, and the 2.6-kb species was found in the heart, kidney and brain, but not in skeletal muscle or liver. The 1.1- and 1.3-kb species were down-regulated in the ventricles of JVS mice, but not in the auricles, and increased to the control level with carnitine treatment. We isolated cDNA clones from ventricle RNA, termed CDV-1 (carnitine deficiency-associated gene expressed in ventricle) and from brain RNA, termed CDV-1R (CDV-1-related gene) by 5′- and 3′-RACE analyses. The entire nucleotide sequence except the 5′-terminal 64 bp of CDV-1 cDNA was completely identical to the 992 bp sequence from the 3′-end of CDV-1R cDNA. The CDV-1 cDNA contained an open reading frame predicting a peptide of 107 amino acids, which composed the C-terminal portion of CDV-1R peptide consisting of 414 amino acids.

Altered Expression of Atrial Natriuretic Peptide and Contractile Protein Genes in Hypertrophied Ventricle of JVS Mice with Systemic Carnitine Deficiency

To characterize cardiac hypertrophy in juvenile visceral steatosis (JVS) mice with systemic carnitine deficiency, we investigated how the hypertrophy develops and whether it is associated with altered expression of any specific genes, especially atrial natriuretic peptide (ANP) and contractile protein genes, in the hypertrophied ventricle. Cardiac hypertrophy in JVS mice became apparent at 10 days after birth and progressed during development. The hypertrophy was observed in the ventricles but not in the atria. ANP mRNA was more intensively expressed in JVS ventricles than in control even at 5 days. Carnitine administration ameliorated the cardiac hypertrophy and suppressed the augmentation of ANP mRNA in the ventricles. Isoform change of expression ofα-actin genes from cardiac to skeletal was seen in the ventricles of JVS mice at 2 weeks. There was no difference in the ratio ofβ-myosin heavy chain mRNA toα-myosin heavy chain mRNA between control and JVS mice at 5 days, but at 2 weeks the ratio was significantly lower in JVS mice than in control. These results suggest that the molecular characteristics of cardiac hypertrophy caused by carnitine deficiency are different from those of cardiac hypertrophy caused by aortic constriction.

Suppressed expression of the urea cycle enzyme genes in the liver of carnitine-deficient juvenile visceral steatosis (JVS) mice in infancy and during starvation in adulthood.

Systemic carnitine-deficient juvenile visceral steatosis (JVS) mice exhibit decreased expression of some liver-selective genes including those for the urea cycle enzymes during the infantile period. At 25 days, carbamoylphosphate synthetase (CPS) mRNA level was remarkably low in the liver of JVS mice, and the HNF-4 and C/EBP-alpha mRNA contents were also reduced. HNF-3 alpha and C/EBP-beta mRNAs were slightly higher in the liver of JVS mice, and HNF-1 mRNA remained normal. These results, together with the developmental changes of these transcription factor mRNA levels, suggest that HNF-4 and C/EBP-alpha are involved in the suppression of CPS expression. If JVS mice survived the crisis at 4-5 weeks, their body weight caught up with that of control mice around 7 weeks. The steady-state levels of CPS and argininosuccinate synthetase (ASS) mRNAs in the liver of JVS mice were normalized by no later than 8 weeks. Starvation for 48 h caused an increase of about twofold in CPS and ASS mRNA levels in the liver of control mice, while the same treatment failed to increase their levels in the liver of JVS mice. The starvation similarly caused increases in HNF-4 and C/EBP-beta mRNA levels in the liver of both control and JVS mice, but the increases were significantly less in JVS mice than in control mice. Thus, the lack of induction of CPS and ASS mRNAs during development and under starvation in JVS mice correlated with the lower induction of HNF-4 and C/EBP-alpha mRNAs, and of HNF-4 and C/ EBP-beta mRNAs, respectively. Furthermore, all these changes seemed to correlate with the presence of fatty liver and the high serum free fatty acid levels, suggesting that disturbance of fatty acid metabolism affects nitrogen metabolism at least in part via altered gene expression of transcription factors such as HNF-4, C/EBP-alpha, and C/EBP-beta.

Mitochondrial abnormalities in a murine model of primary carnitine deficiency. Systemic pathology and trial of replacement therapy.

Mitochondrial abnormalities and effectiveness of replacement therapy were examined in a murine model of systemic carnitine deficiency, namely the juvenile visceral steatosis (JVS) mouse. Homozygous JVS mice revealed severe lipid deposition and abnormal mitochondria in liver, heart, skeletal muscle, and kidney, but there was no pathological change in the nervous system, though they showed cerebral signs. There were numerous ragged-red fibers in muscles, but enzyme activities of the respiratory chain were intact. Histograms of oxidative and nonoxidative muscle fibers showed an increase in small and oxidative muscle fibers in 4-week-old JVS mice, but this difference no longer existed in 8-week- or 1-year-old JVS mice. On the contrary, Mn-superoxide dismutase immunostaining of muscle showed a focal increase in every age of JVS mice. With L-carnitine treatment, JVS mice could survive for a year, but to some extent, there were the same pathological changes as those seen in untreated mice.

Abnormal gene expression causing hyperammonemia in carnitine-deficient juvenile visceral steatosis (JVS) mice.

Autosomal recessive juvenile visceral steatosis (JVS) was discovered in C3H.OH mice in 1985 by Hayakawa et al. (1, 2). The mutant mice showed symptoms very similar to those of Reye’s syndrome, such as fatty liver, hyperammonemia, hypoglycemia and growth retardation. About 70% of the mice died at about 30 days of age. From the symptoms, carnitine deficiency was postulated and verified by analysis of serum and organs. Concentrations of both free and acylcarnitine were found to be very low (10 to 20% of the controls) in every organ examined (3). No abnormal carnitine derivatives were detected in the urine. These results together with the therapeutic effect of carnitine (4) indicate that the mice are a novel animal model of primary systemic camitine deficiency. Thereafter, careful observation of JVS mice revealed cardiac hypertrophy (5), the most typical symptom in the human disease (6). Further studies showed that the primary defect is most probably in the renal camitine transport system (7) and that the gene, jvs, is located on chromosome 11 (8). We analyzed the urea cycle enzymes to investigate hyperammonemia in JVS mice and found its pathogenesis novel and interesting (9). On the other hand, hyperammonemia is one of the symptoms in several inherited diseases of fatty acid metabolism (6, 10–12) and its pathogenesis in relation to urea cycle is not known. JVS mice can provide a means to solve the problem.

Long-chain fatty acids suppress the induction of urea cycle enzyme genes by glucocorticoid action

In order to test the possibility that free fatty acids are the mediator of the abnormal expression of urea cycle enzyme genes in carnitine-deficient juvenile visceral steatosis (JVS) mice, the effects of fatty acids on urea cycle enzyme, carbamoylphosphate synthetase (CPS) and argininosuccinate synthetase (ASS), mRNA levels were examined in rat primary cultured hepatocytes. Addition of a synthetic glucocorticoid hormone, dexamethasone, caused increases in CPS and ASS mRNAs. Further addition of oleic acid suppressed the induction of CPS and ASS mRNAs by dexamethasone. In contrast, the phosphoenolpyruvate carboxykinase (PEPCK) mRNA level induced by dexamethasone was enhanced in the presence of oleic acid. The effects were reversed on further addition of carnitine. The mRNA levels of these enzymes induced by dibutyryl cAMP were not affected by the addition of oleic acid. A study of the specificity of fatty acids revealed that long-chain fatty acids of more than 16 carbons chain length had a suppressive effect on the CPS mRNA level induced by dexamethasone and that the presence of double bonds enhanced the effect. The changes in gene expression of CPS, ASS and PEPCK caused by the fatty acids in the cultured hepatocytes were very similar to those observed in the liver of JVS mice. The AP-1 DNA binding activity in the presence of dexamethasone was slightly enhanced by the addition of oleic acid. These results suggest that the long-chain fatty acids not metabolized in JVS mice are mediators of the abnormal gene expression in the liver which results in hyperammonemia.

Carnitine Transport Defect in Fibroblasts of Juvenile Visceral Steatosis (JVS) Mouse

Juvenile visceral steatosis (JVS) mice are associated with systemic carnitine deficiency (Kuwajima, et al., 1991). In order to investigate the cause of this deficiency, we compared fibroblast carnitine transport activities in normal mice and JVS mice. The kinetic analysis showed that in normal fibroblasts, the Km and Vmax values for saturable uptake was 15.6μM and 2.56 pmol/min/mg protein, respectively. In JVS fibroblasts, however, saturable uptake was not observed. There was no great difference in the linear component of uptake between normal and JVS fibroblasts. At the physiological concentration (50 μ M) of carnitine, the fibroblast carnitine transport activity in JVS mice was decreased to 18 % of that in the normal mice. Thus there is hardly any carnitine transport activity in the fibroblasts of JVS mice, indicating that the JVS mouse can be regarded as an animal model of primary carnitine deficiency.

Disordered expression of glycolytic and gluconeogenic liver enzymes of juvenile visceral steatosis mice with systemic carnitine deficiency

A quantitative study of the effect of carnitine deficiency on expression of glycolytic and gluconeogenic enzymes was performed using juvenile visceral steatosis mice which are systemically deficient in carnitine. The amounts of glucokinase and L-type pyruvate kinase mRNA were reduced in homozygotes, compared to heterozygotes and normal controls at 2 and 8 weeks. Liver-type phosphofructokinase, however, did not differ significantly. The abundance of fructose 1,6-bisphosphatase mRNA was unchanged at 2 and 8 weeks. The level of phosphoenolpyruvate carboxykinase mRNA was increased slightly at 2 weeks, but not at 8 weeks. A part of these changes could not be explained by the plasma glucose or insulin level. Carnitine administration restored the mRNA of these enzymes to normal levels. These results suggest that carnitine deficiency affects the expression of these liver enzymes.

Definition of the Locus Responsible for Systemic Carnitine Deficiency within a 1.6-cM Region of Mouse Chromosome 11 by Detailed Linkage Analysis

Carnitine is an essential cofactor for oxidation of mitochondrial fatty acids. Carnitine deficiency results in failure of energy production by mitochondria and leads to metabolic encephalopathy, lipid-storage myopathy, and cardiomyopathy. The juvenile visceral steatosis (JVS) mouse, an animal model of systemic carnitine deficiency, inherits the JVS phenotype in autosomal recessive fashion, through a mutant allele mapped to mouse chromosome 11. As a step toward identifying the gene responsible for JVS by positional cloning, we attempted to refine the jvs locus in the mouse by detailed linkage analysis with 13 microsatellite markers, using 190 backcross progeny. Among the 13 loci tested, 5 (defined by markers D11Mit24, D11Mit111, D11Nds9, D11Mit86, and D11Mit23) showed no recombination, with a maximum lod score of 52.38. Our results implied that the jvs gene can be sought on mouse chromosome 11 within a genetic distance no greater than about 1.6 cM.

Increased expression of carnitine palmitoyltransferase I gene is repressed by administering L-carnitine in the hearts of carnitine-deficient juvenile visceral steatosis mice.

The juvenile visceral steatosis (JVS) mouse is a novel mutant animal for studying systemic carnitine deficiency. The importance of the model has been pointed out in carnitine-deficient cardiac hypertrophy, since cardiomyopathy has been often improved after oral carnitine therapy in human systemic carnitine deficiency. To understand the effects of carnitine deficiency on gene expression in the heart, we tried to find the genes regulated by carnitine by means of a modified differential display procedure. Carnitine palmitoyltransferase I (CPT I) was one of the isolated genes. The level of CPT I gene expression in the ventricles of the JVS mice was at least three- to sixfold that of normal mice as judged by reverse transcription-polymerase chain reaction (RT-PCR). When the JVS mice were treated with carnitine, CPT I gene expression was repressed to the level of normal mice. Therefore, the increased expression of the CPT I gene was associated with carnitine deficiency.